A new flavoprotein class has emerged within the pyridine nucleotide:disulfide oxidoreductase family. To date two flavoprotein peroxide reductases, the NADH peroxidase and NADH oxidase from Streptococcus faecalis, have been described. The key distinction between these enzymes and disulfide reductases such as glutathione reductase (GR) lies in the unusual cysteine-sulfenic acid (Cys-SOH) redox center, which replaces the redox-active disulfide found in GR. A major goal of this proposal will involve the application of structural, functional, and genetic approaches in order to elucidate a) the structure of the Cys-SOH redox center and those elements of protein structure which contribute to its stabilization, b) the structural basis for the functional divergence between NADH peroxidase (H2O2 yields 2H2O) and NADH oxidase (O2 yields 2H2O), and c) those modular elements of protein structure which distinguish the disulfide and peroxide reductases. The kinetic mechanisms of NADH peroxidase and NADH oxidase will also be probed further through a combination of steady-state and rapid-reaction techniques. In this light, the oxygen reactivity of NADH oxidase provides the potential for significant, new contributions to our understanding of flavoprotein oxygen reactivity. We will also focus our efforts on the streptococcal alpha- glycerophosphate oxidase, which appears to represent one of only two flavoprotein oxidases (O2 yields H2O2) functioning via a hydride transfer mechanism in substrate dehydrogenation. A major goal will be to determine the structural relationship to the two well-defined oxidase classes (the alpha-hydroxy- and alpha-amino acid oxidases) and to the aerobic alpha-glycerophosphate dehydrogenase of Escherichia coli. The information to be gained should be significant not only to the understanding of hydride versus carbanion mechanisms in flavoprotein catalysis, but also to the distinction between flavoprotein oxidase and dehydrogenase functions. The streptococci are heme-deficient facultative anaerobes which rely entirely on substrate-level phosphorylation for ATP synthesis. The roles of these flavin-linked enzymes in streptococcal oxidative metabolism, and their contributions to streptococcal proliferation, are intrinsically linked to the involvement of these organisms in human disease states such as rheumatic fever and endocarditis.